T cell therapy for treatment of viral infections in immune compromised patients holds great promise to be a far superior alternative to conventional anti-viral drugs. After years of work, the last remaining barrier is to eliminate genetic engineering that relies on infectious agents when producing the therapeutic T cell doses. These infectious agents create tremendous complexities and are potentially dangerous to patients. This program will overcome that problem and pave the way for the first off-the-shelf virus-specific T cell therapy in history. At the end of this program the optimal manufacturing specifications for virus specific T cell therapy (VST) directed at adenovirus (AdV), BK, and cytomegalovirus (CMV) infections in immune compromised patients. This will serve an unmet medical need, given there are no FDA approved drugs for AdV and BK, and the only FDA approved drug for CMV is highly toxic and often ineffective. Preliminary data paint a compelling picture of how VST can treats the root cause of viral infection; that is the lack of a functional immune system. Data demonstrate VST can restore the immune system's ability to fight viral infection and has minimal side effects. Preliminary data also show how the manufacturing process that creates VST can be greatly simplified with a novel approaches to virus-specific T cell culture. The specific aims follow up on that data. Aim1 Objective: WWM will construct 6-well G-Rex T cell culture devices, which will allow comparison of multiple culture conditions in parallel and facilitate the rapid identification of key variables that support optimal VST production. Aim 1 Deliverable: We will have fully functional, sterile, 6-well G-Rex devices for the VST optimization studies outlined in Aim 2. The construction of this cell culture tool will allow or collaborators from Baylor College of Medicine to rapidly and efficiently assess multiple conditions in parallel. Aim 2 Objective: Find the optimal stimulation protocol to maximize the purity and killing capacity of a therapeutic VST dose. Aim 2 Deliverable: Optimized virus-specific T cell stimulation protocols that maximize purity and killing capacity will be available for evaluation at full scale in Aim 4. Aim 3 Objective: Use the data of Aim 2 to create G-Rex devices of optimal size for use in the verification study of Aim 4. Aim 3 Deliverable: Full scale G-Rex devices will be available for use in Aim 4. Aim 4 Objective: Verify protocols optimized in Aim 2 are repeatable in full scale G-Rex devices. Aim 4 Deliverable: Specifications defining the most efficient manufacturing conditions for maximum VST killing capacity within alloreactivity acceptance criteria, without the need for infectious agents in the manufacturing process, will be available for use in Phase IIb.